The results of nitric oxide (NO) infusions in normal volunteers and NO infusions and inhalation in experimental animals confirms that NO can be transported as a hormone and thus has the potential to be a pharmacological agent (i.e., a drug). We believe that the lack of vascular effects in our sickle cell patients is due to the presence of circulating hemoglobin and that this contributes to the pathophysiology of this and other chronic and acute hemolytic syndromes, especially the pulmonary hypertension complications which we have found to be severe and of high frequency in older patients. We have analyzed the effects of methemoglobin formation on blood pressure and other cardiovascular parameters in dogs to see if the ferric species of hemoglobin-which could be pharmacologically effected- is safe as has been assumed. To our suprise we find that infusions of methemoglobin lead to prolonged increases in blood pressure and systemic resitance and believe that the mechanisms relates to reduction of methemoglobin to ferrous hemoglobin by plasma ascorbate and the destruction of plasma or cellular NO and nitrite by this species. We also find that NO-bioactivity destruction appears to occur in tissues to a much greater extent than in the vascular bed. An alternative approach to mitigate the effects of cell-free hemoglobin is the infusion of haptoglobin or other agents which may bind hemoglobin or some of its degradation products. Studies of these pathways, in animals and possibly patients, are now being planned. We have recently resumed our work on platelet and blood clotting inhibition by nitrite and are studying the effects of changes in ambient oxygen levels on the processes in rodent models using the TEG methodologies. In recent work we have demonstrated that nitrate ions can be converted to nitrite (and then NO) or to ammonia by various commensal bacteria, depending on oxygen concentration and pH. The half dozen species of microorganisms that we have been studying, especially related to the Lactobacilli, are prevalent in the human oral cavity and gut. We believe that these reactions are important in determining overall nitrogen metabolism in humans and especially the roles of nitrite, NO, and perhaps even nitrate ions in regualting the cardiovascular system. They may also be relevant for the production of ammonia in humans normally and in disease, such as with liver failure. Our second major new project has been to measure nitrate and nitrite levels in various rodent organs. The most surprising result was our finding of very high nitrate levels in muscle tissue. These levels are much higher than in blood and suggest either active transport into muscle or some production of NO and oxidation to nitrate in the tissue itself. Our preliminary results suggest that it is the latter process, with production via NOS1 (nNOS) and oxidation due to reaction with oxymyoglobin, that explains these observations. Most excitingly we have found that exercise, which can increase blood flow 10- or 20-fold, causes marked fall in nitrate levels and transient increases in muscle nitrite levels. We postulate that muscle tissue has a specific mechanism for nitrate reduction, possibly using xanthine oxidoreductase and this process may explain the long mysterious mechanism by which a muscle-produced agent caused marked changes in organ blood flow.